Project Summary/Abstract The human body contains hundreds of thousands of unique small regulatory RNAs that play a substantial role in shaping human gene expression. The majority of these small RNAs can be classified into one of three types: microRNAs (miRNA), small interfering RNAs (siRNA) and piwi-interacting RNAs (piRNA). All three types of small RNA function as guides for members of the Argonaute protein family, which use the encoded sequence information to identify genes and transcripts targeted for silencing. Therefore, the structure and dynamics of the Argonaute proteins dictate the base pairing requirements for target recognition by small RNAs. Importantly, Argonautes do not necessarily require perfect complementarity to their small RNA guide to recognize a target, and the three types of small RNAs are used by the Argonautes in divergent ways. Consequently, understanding and predicting how small RNAs find their targets remains a substantial outstanding challenge with broad implications for understanding regulation of human gene expression. The objective of the proposed research is to provide fundamental insight into the unique features underlying target recognition by the three small RNAs families. Our approach is to leverage Argonaute structure to inform biochemical experiments aimed at identifying modes of target recognition. In Aim 1 we propose to establish criteria for identifying non-conical targeting by miRNAs, which is currently difficult to predict and yet makes up a substantial portion of miRNA:target interactions in cells. Results are expected to enable miRNA target prediction and thereby facilitate researchers studying diverse aspects of human gene regulation. In Aim 2 we will define the conformational switch that Argonaute undergoes in order to transition from using miRNA to siRNA silencing mechanisms. Results are expected to inform siRNA design and enable those harnessing siRNAs to treat patients. In Aim 3 we will determine rules for target recognition by piRNAs, the largest and most poorly understood class of human small RNAs. Expected results will provide fundamental knowledge necessary for deciphering the role of piRNAs in regulation of human gene expression.